The present invention provides means for the detection of hydrolytic enzymes and of polymer-forming enzymes. Elastase is an example of a hydrolytic enzyme. It is an endopeptidase which will digest or cleave a wide variety of protein substrates. It is conventionally assayed by any of several methods involving the measurement of the amount of insoluble elastin substrate solubilized by enzyme digestion. The most convenient assays are those which involve the colorimetric determination of the amount of dye released into solution from digestion of dyed elastin substrates such as Congo Red-elastin, azoelastin, and orcein-elastin.
The Congo Red-elastin method of Naughton and Sanger is a use of fibrin dyed with Congo Red to measure general proteolytic activity. This procedure involves the use of a thoroughly dyed preparation of the substrate elastin. The colored solution that results from the reaction must be separated and subsequently measured, since it is the same color as the starting material. It would be desirable to provide a different means for detection, whereby, as in the present invention, a positive result produces a visibly detected color change not requiring separation.
Glucosyltransferase (GTF) is an example of a polymer-forming enzyme: it catalyzes the synthesis of water-soluble and water insoluble long chain polymers from the substrate sucrose (Buchan et al., Dibdin and Shellis). One method of detecting this enzyme is by using radioactively-labeled sucrose. Reacting this with GTF, any water soluble polymer product which forms is adsorbed onto filter paper separate from the sucrose substrate. The amount of radioactivity incorporated into polymer is measured by conventional scintillation counting. Water insoluble polysaccharide product is measured instrumentally by the measurement of turbidity in a clear solution containing sucrose and the enzyme, or alternatively by measuring the incorporation of radioactivity into polymer which adheres to the sides of an inclined glass test tube containing radiolabeled sucrose and glucosyltransferase.
There are various agglutination assays that use a flocculating colloid to indicate the result. One example is the polystyrene latex agglutination test. An aggregation of polystyrene latex colloidal particles occurs when adsorbed or coupled antibody attached to the particles' surface reacts with antigen in an aqueous sample (U.S. Pat. No. 4,164,558). Another example (U.S. Pat. No. 4,313,734) employs colloidal metal sols to produce a color change. However, these agglutination assays are based on ligand binding and do not provide a means for detecting an enzyme.
Using bovine serum albumin (BSA) and gelatin, a melted, denatured form of collagen, and Congo Red, a colloidal dyestuff, I discovered that high concentrations of BSA or gelatin tend to protect colloidal dyes from flocculation by a concentration of simple salts-sodium or potassium chloride-which ordinarily would have flocculated had the BSA or gelatin not been added. This is an example of colloidal protection.